Soluble ALDC derivative and use thereof

ABSTRACT

The ALDC derivative is produced by treating ALDC in an aqueous medium with glutaraldehyde in defined proportions with optional subsequent immobilization. The ALDC derivative which is used in beer fermentation exhibits a satisfactory stability at low pH.

The invention comprises a soluble ALDC derivative and a use thereof.ALDC is an abbreviation for acetolactate decarboxylase.

By fermentation of carbohydrate containing substances, e.g. wort orgrape juice, various by-products may be formed by processes other thanthe wanted alcoholic fermentation. Thus, an unwanted by-product isdiacetyl.

It appears from EP 46066 that ALDC can be used as an enzyme, whichprevents the formation of diacetyl. However, the pH optimum of mostnaturally occurring ALDC's is around 6, and the activity at pH 3.8-4.7,which is the pH of the fermenting wort, is too low for practicalpurposes, especially at pH below 4, which is a usual pH of fermentingworts with low malt content. For that reason, the process of preventingthe formation of diacetyl from fermenting beer or wine has not yet foundits way into the practical industrial production on any larger scale. InEP 46066 it is stated that ALDC may be chemically modified to shift theoptimum activity to lower pH values, reference being made to Biochem.,vol. 11, No. 22, 1972 (p. 4022-4084). The modification methods mentionedin the Biochem. article comprise growing poly(ornithyl) side chains onchymotrypsin; the Biochem. article thus does not offer any suggestionsfor modification of ALDC. The prior art modification method for shiftingthe optimum activity to lower pH values is not suitable for industrialapplication, and furthermore it is not known, if the poly(ornithyl)method can be transferred from chymotrypsin to ALDC. Finally, it doesnot appear from the Biochem. article, if the stability at low pH of theprior art modified chymotrypsin is satisfactory.

Thus, the purpose of the invention is the provision of an ALDCderivative which can be used with advantage in industry, and whichexhibits a satisfactory stability at low pH.

Surprisingly, it has been found that treatment of ALDC withglutaraldehyde provides a soluble modified ALDC, which in the firstplace exhibits the wanted pH profile, which in the second place is cheapand easily manufactured, and which in the third place exhibits asatisfactory stability.

Thus, the soluble ALDC derivative according to the invention ischaracterized by the fact that ALDC in an aqueous medium is treated withglutaraldehyde in a concentration corresponding to between 0.1 and 5 gof glutaraldehyde per g of pure ALDC protein, preferably correspondingto between 0,25 and 2 g of glutaraldehyde per g of pure ALDC protein. Ithas been found that this ALDC derivative is soluble, if not furthertreated and that it can be produced in a high activity yield.

In Biotechnology Letters Vol. 10 No. 5 325-330 (1988) it is describedthat crosslinking of β-glucosidase with glutaraldehyde provides aderivative with improved thermal stability. In Adv. Biochem. Eng. 12, p.41-118, 1979, Rolf D. Schmid describes that crosslinking of differentenzymes, e.g. papain, glucose oxidase, catalase and uricase, but notALDC, provides enzyme derivatives with improved thermal stability. Thus,the prior art does not point to glutaraldehyde as an agent which couldfulfil the purpose of the invention.

It is to be understood that all ALDC enzymes, i.e. ALDCs produced fromany microorganism, can be used according to the invention. PreferredALDC's are from Bacillus brevis and Bacillus licheniformis.

Reference can be made to EP 131251, which describes a special inorganiccarrier, on which enzymes, e.g. ALDC, are immobilized by adsorption andcrosslinking with glutaraldehyde. The invention, in contradistinctionthereto, comprises a soluble ALDC derivative exclusively. Also this EP,does not describe the shift of the pH of the pH-activity curve of theALDC, which is one of the main features of the present invention.Another main feature of the present invention, which is not described inthe EP, is the treatment of the ALDC with glutaraldehyde in a definedsmall concentration.

Also, the invention comprises a use of the soluble ALDC derivativeaccording to the invention in beer fermentation.

A preferred embodiment of the use according to the invention ischaracterized by the fact that the soluble ALDC derivative is usedtogether with ordinary yeast in a batch fermentation. This is thesimplest way of using the soluble ALDC derivative according to theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the activities of the ALDC derivative ofthe present invention (derivatized ALDC) and normal ALDC at differentpHs.

FIG. 2 shows a comparison of the residual activities of derivatized ALDCand normal ALDC after contact with pasteurized beer over a period ofseveral days at pH 4.

FIG. 3 shows a comparison of the residual activities of derivatized ALDCand normal ALDC after contact with pasteurized beer over a period ofseveral days at pH 6.

FIG. 4 shows the pattern of pH change in a traditional batchfermentation, using brewery malt wort, with or without varyingconcentrations of sucrose, over a period of seven days.

FIG. 5 shows the pattern of residual ALDC activity remaining in atraditional batch fermentation, using brewery malt wort, with or withoutvarying concentrations of sucrose, over a period of seven days.Pr.1=normal ALDC preparation; Pr.2=derivatized ALDC.

FIG. 6 shows a comparison of the change in total diacetyl in atraditional batch fermentation, using derivatized ALDC or normal ALDCwith brewery malt wort alone, over a period of seven days. Prep.1=normalALDC preparation; Prep.2=derivatized ALDC.

FIG. 7 shows a comparison of the change in total diacetyl in atraditional batch fermentation, using derivatized ALDC or normal ALDCwith brewery malt wort plus 10% sucrose, over a period of seven days.Prep.1=normal ALDC preparation; Prep.2=derivatized ALDC.

FIG. 8 shows a comparison of the change in total diacetyl in atraditional batch fermentation, using derivatized ALDC or normal ALDCwith brewery malt wort plus 20% sucrose, over a period of seven days.Prep.1=normal ALDC preparation; Prep.2=derivatized ALDC.

FIG. 9 shows a comparison of the change in total diacetyl in atraditional batch fermentation, using derivatized ALDC or normal ALDCwith brewery malt wort plus 40% sucrose, over a period of seven days.Prep.1=normal ALDC preparation; Prep.2=derivatized ALDC.

The invention will be illustrated by means of the following examples. Inthese examples an ALDC preparation will be used, produced by cultivationof a Bacillus subtilis strain containing a gene encoding and expressingthe ALDC of Bacillus brevis with properties described in DK 149335B.

EXAMPLE 1

100 ml of ALDC solution (batch EDF 212, which is the fermentation liquorcentrifugate with an ALDC activity of 1700 ADU/ml) was mixed with 2.5 mlof a 2% glutaraldehyde solution (final glutaraldehyde concentration0.05% (w/w, corresponding to 0.5 g of glutaraldehyde/g of ALDC). Thereaction mixture was cooled with ice for three hours. pH was constantlyadjusted to 7.5.

The ALDC activity (ADU/ml) was measured by Novo Analysis method AF278/1-GB (available on request from Novo Nordisk A/S, Novo Alle, DK-2880Bagsvaerd) with pH adjusted in the substrates to values ranging from 3.6to 6.0. The pH-activity curves are shown in FIG. 1.

The stability of ALDC and the derivatized sample was measured in anormal, pasteurized Danish beer (HOF). 20 ADU was added per ml beer inwhich pH was adjusted to 4.0 and 6.0, respectively. Samples from thebeers were taken each day, and the residual ALDC activity measured at pH6.0. The results are shown in FIGS. 2 and 3.

It is concluded that treatment with glutaraldehyde improves both theactivity and the stability at pH 4.0 of ALDC.

EXAMPLE 2 Testing of Derivatized ALDC in a Traditional BatchFermentation

ALDC (batch EDF 212, 1700 ADU/ml) (preparation 1) and a glutaraldehydetreated ALDC (preparation 2) prepared as described in Example 1 (1100ADU/ml) were used.

The fermentations were carried out with brewery malt wort (FAXE) and thesame wort with added amounts (10, 20, 30 and 40% w/w) of adjunct(sucrose). 2.5 g of yeast was added per liter of wort. ALDC was addedtogether with the yeast (46 ADU per liter). The fermentations werecarried out at 12° C. and lasted for 7 days. pH (FIG. 4), ALDC residualactivity (FIG. 5) and total diacetyl (α-acetolactid acid+diacetyl)(FIGS. 6-9) were measured each day.

The results show that the reduction of total diacetyl is larger when thederivatized ALDC (prep. 2) is used than when untreated ALDC is used, andthat the derivatized preparation is more stable.

I claim:
 1. A soluble derivative of acetolactate decarboxylase (ALDC),wherein the derivative is obtained by treatment of ALDC in an aqueousmedium with glutaraldehyde, in a concentration of between 0.1 and 5 g ofglutaraldehyde per g of pure ALDC protein in the medium.
 2. Thederivative of claim 1, wherein the glutaraldehyde is in a concentrationof between 0.25 and 2 g of glutaraldehyde per g of pure ALDC protein. 3.A method for removing diacetyl from a beer fermentation liquorcomprising contacting the fermentation liquor with a soluble ALDCderivative obtained by treatment of ALDC in an aqueous medium withglutaraldehyde in a concentration of between 0.1 and 5 g ofglutaraldehyde per g of pure ALDC protein in the medium.
 4. The methodof claim 3, which is conducted with non-immobilized yeast in a batchfermentation.